Type I collagen is the most abundant protein in higher vertebrates. It forms fibrous scaffolds of bone, skin, vasculature, and other tissues. Mutations in type I collagen typically result in Osteogenesis Imperfecta (OI), Ehlers-Danlos syndrome (EDS) or a combination of OI and EDS. Most known pathogenic mutations are substitutions of an obligatory glycine in the repeating Gly-X-Y triplets of the collagen triple helix. It is generally accepted that disruption of the triple helix folding and structure by these mutations is somehow involved in the disease, but no clear relationship between different substitutions and rather dramatic variations in the disease severity associated with them has been found so far. We have established that the effect of Gly substitutions on the overall collagen stability depends on their location within different regions of the triple helix but not on the identity of the substituting residues. These regions appear to align with regions important for collagen fibril assembly and ligand binding as well as with some (but not all) of the observed regional variations in OI phenotypes. In an ongoing study of different substitutions, we are continuing mapping of these regions and analysis of their association with OI phenotype variations. Substitutions at the X or Y position in the triple helix, mostly of cysteine for arginine, were recently identified in individuals with OI, osteopenia, arterial rupture, EDS, and Caffey disease. Since these non-glycine substitutions were not considered to be detrimental for the triple helix folding and structure, their molecular pathophysiology remained completely unclear. In 2007, we reported a change in the triple helix stability caused by a Cys substitution for an Y position Arg-888 in several members of the same family affected with a combination of OI and EDS symptoms. During the past year, we described a similar change in the triple helix stability in unrelated patients, in which Arg-780 in the alpha-1 chain was substituted with Cys or Leu. This study confirmed the role of Y-position Arg residues in the triple helix stability proposed by several authors. It suggested that the OI phenotype of patients with Y-Arg substitutions might be caused by the destabilizing effect of an Y-Arg loss rather than by the gain of a Cys residue not normally present in the triple helix. At the same time, analysis of our and literature data indicated that the Cys gain might be responsible for the arterial rupture and EDS phenotypes. We observed formation of aberrant disulfide bonds between the Cys residues in molecules with two mutant alpha-1 chains. These bonds resulted in kinking, register shift, and abnormal N-propeptide cleavage from the triple helix, the latter effect known to be involved in EDS. In addition to characterization of molecular defects in OI and EDS patients, we continued studies of murine OI models with knocked-in Gly substitutions. In particular, we utilized Raman microspectroscopy to characterize defects in organic matrix composition and bone mineralization in mice with a Cys substitution for Gly-610 in the alpha-2 chain and in mice with a Cys substitution for Gly-349 in the alpha-1 chain of type I collagen. These studies revealed significantly reduced collagen content and hypermineralization of the matrix, likely contributing to the bone brittleness in these animals observed in biomechanical measurements. In the Gly-349-Cys mice, we investigated the effect of intrauterine transplantation of bone marrow stromal cells on the disease progression. One of the major challenges in the stem cell therapy is low engraftment of donor cells. However, our study revealed that the donor cells comprising just 2% of osteoblasts were responsible for synthesis of about 20% of bone matrix and dramatic improvement in the matrix composition and mineralization, eliminating 30% perinathal lethality associated with this mutation in mice and nearly normalizing biomechanical properties of bones in adult animals. One of our most significant advances in the past two years was the characterization of a collagenase-resistant isoform of type I collagen and its potential role in cancer, fibrosis, and other disorders. The normal isoform of type I collagen is a heterotrimer of two alpha-1 and one alpha-2 chains. However, homotrimers of three alpha-1 chains were found in some carcinomas, fibrotic tissues, and rare forms of OI and EDS associated with alpha-2 chain deficiency. Our studies of the homotrimeric collagen from an EDS patient revealed its surprising resistance to cleavage by the most common interstitial collagenase (MMP-1). While pursuing this observation, we discovered that the homotrimers were at least 5-10 more resistant to all major collagenolytic matrix metalloproteinases (MMPs), including MMP-1,2,8,13, and 14. A more detailed investigation revealed this resistance to be related to an increased stability of the homotrimer triple helix at the primary MMP cleavage site, inhibiting unwinding of the helix at this site (necessary for the cleavage). Overexpression of MMPs is a hallmark of invasive cancers;cleavage of stromal type I collagen fibers by cancer cells and recruited fibroblasts is an essential step in clearing an invasion path for the cancer. Therefore, we hypothesized that synthesis of MMP-resistant collagen isoform may support cancer cell proliferation and tumor invasion;the MMP-resistant fibers laid down by these cells may serve as tracks, supporting outward cell migration and tumor growth. Our measurements in cell culture and xenograft tumor models confirmed the synthesis of a significant fraction of the homotrimers by cancer cells (20-40% in culture and even larger in vivo) but no homotrimer synthesis by normal mesenchymal cells or fibroblasts recruited into tumors. Furthermore, we found that more rigid homotrimeric type I collagen matrix supported faster proliferation and migration of cancer cells. Presently, we are investigating how the homotrimer vs. heterotrimer synthesis is regulated in different cells and developing approaches to selective targeting of the homotrimers. Since the homotrimers do not appear to be produced in normal tissues, they may present a novel, appealing diagnostic and therapeutic target. In addition to carcinomas, type I collagen homotrimers were reported in liver fibrosis and other fibrotic disorders. Since collagen fiber degradation by MMPs is essential for preventing fibrosis, it was reasonable to assume that MMP-resistant isoform of type I collagen would be involved in the disease and would be a common factor in different forms of fibrosis. In the past year, we found that the homotrimers were indeed involved in glomerular sclerosis in at least one murine model. However, further testing revealed no homotrimers in tissue biopsies from scleroderma or uterine fibroma patients. Based on these observations, our experience gained in the studies of cancer models, and literature reports, we now hypothesize that the homotrimers are not produced by normal or activated mesenchymal cells but only by non-mesenchymal, undifferentiated, dedifferentiated, or transformed cells. While we still believe that the homotrimers may play an important role in some forms of fibrosis, they are not likely to be involved in fibrosis within mesenchymal tissues.